Figure 6. K50 motifs are required for expression in both photoreceptors and bipolar cells, while Q50 motifs mediate repression of reporter expression specifically in bipolar cells.

(A) Schematic of reporter construct containing 820 bp from the promoter region and 5’ untranslated region (UTR) of mouse Gnb3. This region contains two phylogenetically conserved blocks harboring a total of five K50 motifs (black ellipses). (B) Left, Schematics of reporter pairs containing wild-type (WT; black) and inactivated (crossed out) K50 motifs. Right, Each pair of reporters was electroporated into explanted newborn mouse retina, which were subsequently harvested after eight days in culture and photographed in a flat mount preparation. While K50 sites 2, 4 and 5 are required for reporter expression, sites 1 and 3 are dispensable. (C) Left, Pairs of reporters containing WT, inactivated K50, or novel Q50 (TAATTA; yellow) motifs were electroporated into newborn mouse retina in vivo. After 20 days, the retinas of electroporated mice were harvested and photographed in vertical cross-sections. (C) Right, Representative cross-sections of retinas injected with the indicated pair of reporters. Loss of both K50 motifs 1 and 3 has no effect on expression in either photoreceptors or bipolar cells, while converting these same sites to Q50 motifs abrogates expression specifically in bipolar cells (n = 2–7 depending on reporter pair, Figure 6—source data 1). White arrowheads indicate bipolar cells expressing the WT reporter, but not the mutated one. (D) Table summarizing the results of reporter analysis presented in (B) and (C).